The present invention relates generally to mass spectrometry and more particularly to systems and methods for improving the analysis of chromatographic peaks using a tandem mass spectrometer.
Mass spectrometry (MS) is an analytical technique used to measure the mass-to-charge ratio (m/z) of ions. A mass spectrometer is a device used for mass spectrometry, and produces a mass spectrum of a sample to find its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. A typical mass spectrometer comprises three parts: an ion source, a mass analyzer, and a detector.
Tandem mass spectrometry involves two or more stages of mass selection or analysis, usually separated by a stage of fragmentation. A tandem mass spectrometer is capable of multiple rounds of mass spectrometry. For example, in a first stage, one mass analyzer can isolate one precursor compound ion from many entering a mass spectrometer. The compound ions can then be fragmented in a second stage which may include a collision cell. Compound ions are typically confined to the collision cell, stabilized via a multipole, and fragmented via collision-induced dissociation (CID) with inert gas molecules. A second mass analyzer then separates the fragment ions produced from the compound ions, and the fragment ions are detected using a detection system. The result is a mass spectrum of the fragment ions for each compound ion.
The compound ions may be introduced into the first mass analyzer concurrently and over a limited time frame, such as across a liquid chromatographic (LC) peak. During LC/MS analysis of complex samples, e.g. the trypsin-digested protein content of human serum, hundreds to thousands of compounds may be present. Even when operating with separation regimes delivering high peak capacity, at any particular point in time, multiple compounds are typically eluted in parallel. Thus, multiple co-eluting compounds must be analyzed within the same time window, which can make obtaining sufficient and accurate data difficult.
Also, chromatographic peaks may be narrow. For example, Agilent's new HPLC-Chip increases chromatographic resolution by creating narrow chromatographic peaks (usually 2-3 seconds wide). The narrow peaks give less time to sample the compound ions, but give a greater abundance of the compound ions. The combination of multiple co-eluting compounds and narrow chromatographic peaks requires a fast sampling rate for MS/MS analysis to be successfully applied. For instance, to perform an MS/MS analysis (one cycle) of co-eluting compounds A, B and C over a three second elution window, on average only one second can be used to analyze each of the corresponding compound ions.
The analysis of each co-eluting compound could occur during a one second sub-cycle, giving three sub-cycles. However, one may not know exactly where in the chromatographic peak (elution window) that a specific co-eluting compound, e.g. A, is concentrated. Thus, an insufficient amount of compound ion A could be measured if A is concentrated at the end of the elution window and its analysis sub-cycle occurs at the beginning of the elution window.
To improve coverage, typically a first mass analyzer cyclically measures each of the three ions multiple times across the elution window. In this manner, the likelihood of measuring compound A is increased. A lack of fast switching often contributes to poor statistical coverage of an elution window due to relatively long and contiguous blocks of time that a compound ion is not measured.
Also, during a sub-cycle for a specific ion, the collision energy may be varied over a range. This is typically done, for example, because the ideal collision energy may not be known. Systems and methods have been hampered in not being able to provide accurate measurements at different collision energies of different compound ions across the same elution time window.
Therefore it is desirable to provide systems and methods that overcome the above and other problems.